Production of alkaloids by liliaceae cell culture

ABSTRACT

The invention relates to the production of alkaloids from Liliaceae cell culture.

FIELD OF THE INVENTION

The present invention relates to the production of alkaloids fromLiliaceae cell culture.

BACKGROUND OF THE INVENTION

The Hedgehog (Hh) pathway plays a critical role in human embryogenesisand tissue differentiation, and thus, disruption of the Hh pathway isimplicated in some birth defects as well as cancers. Small molecules canbe used to activate or block the Hh pathway, e.g., by targeting Patched,a protein of the Hh pathway that inhibits cell division, or Smoothened,a protein of the Hh pathway that promotes cell division. For example,the alkaloid cyclopamine, which is found in plants of the Veratrum genusof the Liliaceae family, can block the Hh pathway by targetingSmoothened, thus inhibiting cell growth. Cyclopamine and other alkaloidsof the Veratrum superfamily have been identified as potentialtherapeutic agents to treat diseases in which the Hh pathway isimplicated. Likewise, biosynthetic precursors, derivatives, or syntheticderivatives of such alkaloids may be therapeutically active.

It has long been known that cyclopanine, when ingested by animals,causes severe neural defects, and now its role in tumorigenesis hasgarnered attention. Cyclopamine can block the action of mutated genesthat produce basal cell skin carcinomas, the most common form of humancancer. Studies in mouse cells suggest that cyclopamine may be used totreat a number of cancers, including medulloblastomas in the brain andrhabdomyosarcomas in muscle. Past findings also spotlight the promise ofmechanism-based treatment approaches that target specific signalingpathways that are critical to a particular cancer.

Because cyclopamine and other alkaloids of the Veratrum superfamily mayprove to be useful anti-cancer drugs, there is a need for efficientproduction methods for these compounds. The commercial production ofalkaloids and other secondary metabolites is unpredictable in that evenwhen a plant is known to produce a particular metabolite, it isunpredictable whether the plant cells will produce the metabolite in anundifferentiated cell culture. Ma et al. has disclosed “[a]n in vitroculture system for somatic embryogenesis and green plant regeneration ofVeratrum californicum,” but the Abstracts do not disclose production ofalkaloids from undifferentiated cell culture. Ma et al., “SomaticEmbryogenesis and Green Plant Regeneration from Veratrum californicum,”11^(th) IAPTC&B Congress Poster Sessions P-1033. Ritala et al., “Tissueculture and genetic engineering of an important anticancer compoundproducing plant Veratrum californicum Duran,” Planta Medica 2006; 72.See also U.S. Pub. No. 2009/0305338. Veratrum alkaloids may also beproduced in cultures of differentiated tissue such as shoots or roots.These differentiated tissues may be derived from initiallyundifferentiated tissue or through genetic transformation, e.g., hairyroots or shooty teratomas or the like. Transformed tissue may also becultivated as suspension of undifferentiated cells. The presentinvention provides methods of producing alkaloids in undifferentiatedcell culture.

SUMMARY OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The invention provides for various methods of producing alkaloids from aplant cell culture. One objective of the invention is to obtaincommercially significant amounts of the end product from large volumeaerated fermentors. Another objective is to provide methods ofincreasing the volumetric yield.

In one embodiment, the invention provides for a method of producing oneor more alkaloids from suspension cell culture by culturing plant cellsof the family Liliaceae in a nutrient medium to form a cell culture thatproduces one or more alkaloids of the superfamily of Veratrum alkaloids,or a precursor of such an alkaloid or a derivative of such an alkaloid,and recovering one or more alkaloids.

In some embodiments, the plant cells are Veratrum or Amianthium cells.In a specific embodiment, the plant cells are Veratrum californicumcells. In preferred embodiments, the cells are undifferentiated cells,but not embryogenic cells.

In another embodiment, the plant cells are cultured in a growth medium.In a specific embodiment, the growth medium is capable of inducing agrowth increase of at least 50% in one week. In another embodiment, theplant cells are cultured in a production medium.

In another embodiment, the plant cells are cultured in a growth mediumand then subsequently cultured in a production medium. In a specificembodiment, the growth and the production media are different. Inanother specific embodiment, plant cells of the genus Veratrum arecultured in a growth medium that is capable of inducing a growthincrease of at least 50% in one week, these plant cells are thencultured in a production medium that is different from the growth mediumand that yields at least about 0.1 mg/L of one or more alkaloids of thesuperfamily of Veratrum alkaloids or precursors or derivatives thereof,and then at least one alkaloid is recovered.

In another embodiment, the invention provides for various cultures ofcells such as suspension cultures of Veratrum cells. In a specificembodiment, the invention provides for a culture of Veratrum cells thatis capable of producing at least about 0.1 mg/L of one or more alkaloidsof the superfamily of Veratrum alkaloids or precursors or derivativesthereof. In another embodiment, the invention provides for a suspensionculture having cells of the family of Lilliaceae and one or morealkaloids of the superfamily of Veratrum alkaloids in an amount of atleast 0.1 mg/L. The invention also provides for alkaloids, precursors,derivatives, or extracts of these undifferentiated cell cultures.

In another embodiment, the methods and cultures described herein yieldat least about 0.1 mg/L of one or more alkaloids, more preferably atleast about 0.3 mg/L, at least about 0.5 mg/L, at least about 0.75 mg/L,at least about 1 mg/L, or at least about 1.5 mg/L. These yields can bemeasured as the production of one or more individual alkaloids or as ameasure of total alkaloids.

In another embodiment, the alkaloid contains aC-nor-D-homo-[14(13->12)-abeo] ring. In preferred embodiments, thealkaloid is cyclopamine or jervine.

In another embodiment, the invention provides a method for producing oneor more C-nor-D-homo-[14(13->12)-abeo] ring system-containing alkaloidcompounds where undifferentiated plant cells of a plant belonging to thefamily Liliacaeae, and which produce one or moreC-nor-D-homo-[14(13->12)-abeo] ring system-containing alkaloidcompounds, are cultured in nutrient medium, and theC-nor-D-homo-[14(13->12)-abeo] ring system-containing alkaloid compoundsare recovered from the resulting culture. In other embodiments, cellsare cultured in the presence of biotic and/or abiotic factor(s) whichstimulate and/or promote the biosynthesis of, for example,C-nor-D-homo-[14(13->12)-abeo] ring system containing alkaloidcompounds.

In another embodiment, the invention provides for a method of producinga C-nor-D-homo-[14(13->12)-abeo] ring containing alkaloid compound byculturing cells of a plant belonging to the Liliaceae family, whichproduce a C-nor-D-homo-[14(13->12)-abeo] ring containing alkaloidcompound, in a vessel in the presence of one or more stimulants whichpromote the biosynthesis of the C-nor-D-homo-[14(13->12)-abeo] ringcontaining alkaloid, and where the gas phase in the culture vessel iscontrolled to less than the oxygen concentration in the atmosphere fromthe initial stage of the culture, or wherein the dissolved oxygenconcentration in a fluid medium which is in contact with the cells iscontrolled to less than the saturated dissolved oxygen concentration atthe temperature from the initial stage of the culture, and recoveringC-nor-D-homo-[14(13->12)-abeo] ring containing alkaloid compound fromthe resulting cultures. In another embodiment, the cells of the plantwhich produce the C-nor-D-homo-[14(13->12)-abeo] ring containingalkaloid compound are cultured by introducing oxygenic gas containing0.03%-10% of carbon dioxide into the culture vessel.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, B, and C show the detection of cyclopamine in a Veratrumcalifornicum callus by LC-MS. FIG. 1A shows extracted single ion (m/z412 M⁺+H of cyclopamine). FIG. 1B shows MS spectrum of TIC peak at 3.40,and FIG. 1C shows TIC spectrum of callus extract.

FIGS. 2A, B, and C show the detection of cyclopamine in a Veratrumcalifornicum callus by LC-MS. FIG. 2A shows extracted single ion (m/z412 M⁺+H of cyclopamine). FIG. 2B shows MS spectrum of TIC peak at 3.40,and FIG. 2C shows TIC spectrum of callus extract.

FIG. 3 shows the multiple reactions monitoring (MRM) spectra of Veratrumcell suspension samples. The ions for two analytes of interests weredetected above the noise level: cyclopamine and jervine.

FIG. 4 shows the multiple reactions monitoring (MRM) spectra of aVeratrum californicum suspension culture by UPLC/MS/MS. The sample wasconcentrated before injection.

FIGS. 5A and B show cyclopamine titers in MS- and SH-based productionmedium on days 8, 9, and 10 using a Veratrum californicum suspensionculture. FIG. B shows the data not including outliers from dry wells.

FIG. 6 shows cyclopamine titers in MS- and SH-based production medium ondays 8, 9, and 10 using a Veratrum californicum suspension culture.

FIG. 7 shows cyclopamine titers in MS- and SH-based production medium ondays 8, 9, and 10 using a Veratrum californicum suspension culture.

FIG. 8 shows cyclopamine titers in MS- and SH-based production medium ondays 8, 9, and 10 using a Veratrum californicum suspension culture.

FIG. 9 shows cyclopamine titers in MS- and SH-based production medium ondays 8, 9, and 10 using a Veratrum californicum suspension culture.

DETAILED DESCRIPTION OF THE INVENTION Alkaloids and Alkaloid-ProducingCells

The term “alkaloid” as used herein means any member of the superfamilyof Veratrum alkaloids as well as precursors of such an alkaloid andderivatives of such an alkaloid. In a preferred embodiment, the presentinvention relates to the production of a member of the superfamily ofVeratrum alkaloids. In a preferred embodiment, the present inventionrelates to the production of C-nor-D-homo-[14(13->12)-abeo] ring systemcontaining alkaloid compounds. Examples of Veratrum alkaloids include,but are not limited to, those listed in the table below:

TABLE 1 Exemplary Veratrum Alkaloids Name CAS Reg. No. Cevine 124-98-1Geralbine Germine 508-65-6 Germine; O³-(R)-2-Methylbutanoyl 465-78-1Germine; O¹⁵-(R)-2-Methylbutanoyl 42138-61-4 Germine;O¹⁵-(R)-2-Methylbutanoyl, O³—Ac 465-77-0 Germine;O¹⁵-(R)-2-Methylbutanoyl, O³, 508-66-7 O⁷-di-Ac Germine;O¹⁶-(2-Methylbutanoyl) 135636-53-2 Germine; O³,O¹⁵-Bis(2-methylbutanoyl)175030-77-0 Germine; O³,O¹⁵-Bis(2-methylbutanoyl), O⁷—Ac Germine;O¹⁵-Angeloyl 240802-94-2 Germine; O³-Angeloyl, O¹⁵-(R)-2- 639-11-2methylbutanoyl, O⁷—Ac Germine; O-Angeloyl, O-tigloyl, O—Ac Germine;O³,O¹⁵-Diangeloyl, 7-Ac 122332-72-3 Germine;O³,O¹⁵-Bis(2-methyl-2-butenoyl) 90541-57-4 Germine;O³-(2-Hydroxy-2-methylbutanoyl), 134357-41-8 O¹⁵-(2-methylbutanoyl)Germine; O³-(R)-2-Hydroxy-2-methylbutanoyl, 508-67-8O¹⁵-(R)-2-methylbutanoyl Germine; O³-(R)-2-Hydroxy-2-methylbutanoyl,560-48-5 O¹⁵-(R)-2-methylbutanoyl, O⁷—Ac Germine;O³-(2R,3R)-2,3-Dihydroxy-2- 426-34-6 methylbutanoyl,O¹⁵-(R)-2-methylbutanoyl Germine; O³-(2R,3R)-2,3-Dihydroxy-2- 58162-51-9methylbutanoyl, O¹⁵-(R)-2-methylbutanoyl, O⁷—Ac Germine;O³-(2S,3R)-2,3-Dihydroxy-2- 595-64-2 methylbutanoyl,O¹⁵-(R)-2-methylbutanoyl Germine; O³-(2S,3R)-(3-Acetoxy-2- 23211-84-9hydroxy-2-methylbutanoyl), O¹⁵-(R)-2- methylbutanoyl Germine;O³-(2S,3R)-(3-Acetoxy-2- 465-75-8 hydroxy-2-methylbutanoyl), O¹⁵-(R)-2-methylbutanoyl, O⁷—Ac Germine; O¹⁵-(3,4-Dimethoxybenzoyl) 33352-59-9Germine; O¹⁵-(3,4-Dimethoxybenzoyl), O³—Ac 33352-58-8 Germine;O³-(3,4-Dimethoxybenzoyl), 142735-72-6 O¹⁵-(2-methylbutanoyl) Germine;O³-(3,4-Dimethoxybenzoyl), 214046-03-4 O¹⁵-(2-methylbutanoyl), O⁷—AcGermine; 1α-Acetoxy, O³-(2ξ-hydroxy-2- 182693-36-3 methylbutanoyl),O¹⁵-(2-methylbutanoyl) Hakurirodine 56857-49-9 Hakurirodine; 22R,28-Dihydro 38636-84-9 Hakurirodine; 22R, 28-Dihydro, 3-O—Ac 38636-85-0Hosukinidine 72765-23-2 Isorubijervine 468-45-1 Isorubijervine;O³-β-_(D)-Glucopyranoside 468-46-2 Isorubijervine; 12β-Hydroxy164178-46-5 Jervine 469-59-0 Jervine; O—Ac (O-Acetyljervine) 14788-78-4Jervine; N-Methoxycarbonyl (Verapatuline) Jervine;N-(2-Methoxycarbonylethyl) 132943-48-7 (Methyl jervine-N-3′-propanoate)Jervine; O³-β-_(D)-glucopyranoside 36069-05-3 (Pseudojervine) Jervine;3-Ketone (Jervinone) Jervine; 11β-Alcohol (Veratrobasine) 20226-97-5Jervine; 11β-Alcohol, 3,11-di-Ac Jervine, 11-Deoxo (Cyclopamine)4449-51-8 Jervine, 11-Deoxo, O,N-di-Ac Jervine; 11-Deoxo,3-O-β-_(D)-glucopyranoside 23185-94-6 (Cycloposine) Jervine; 1α-Hydroxy,5α,6-dihydro Loveraine 20-(2-Methyl-1-pyrrolin-5-yl)pregn-4-en-3-one55486-07-2 Neoverataline A Neoverataline A; 7α-Hydroxy Petisidinine;3-Ac Procevine 468-24-6 Protoverine; O⁶,O⁷-Di-Ac, O¹⁵-(R)-2- 143-57-7methylbutanoyl, O³-(+)-2-hydroxy- 2-methylbutanoyl Protoverine;O⁶,O⁷-Di-Ac, O¹⁵-(R)-2- 124-97-0 methylbutanoyl,O³-(+)-threo-2,3-dihydroxy- 2-methylbutanoyl Protoverine; O⁶—Ac,O¹⁵-(R)-2-methylbutanoyl, 67375-42-2 O³-(+)-2-hydroxy-2-methylbutanoylProtoverine; O⁶—Ac, O¹⁵-(R)-2-methylbutanoyl, 67370-03-0O³-(+)-threo-2,3-dihydroxy-2-methylbutanoyl Protoverine; O⁶,O⁷-Di-Ac,O¹⁵-(R)-2- 663-93-4 methylbutanoyl, O³-angleloyl Protoverine;3-[(2S,3R)-2,3-Dihydroxy-2- 82535-71-5 methylbutanoyl], O⁶,O⁷-di-Ac,O¹⁵-(R)-2- methylbutanoyl Protoverine; O³-(2-Hydroxy-2-methylbutanoyl),O¹⁵-(2-methylbutanoyl) Rubijervine 79-58-3 Rubijervine; 12-Epimer472-00-4 Rubiverine 16,28-Secosolanida-5,22(28)-diene-3,16-diol, _(9Cl);29271-49-6 (3β,16α,20S,25α)-form16,28-Secosolanida-5,22(28)-diene-3,16-diol, _(9Cl); 54557-67-4(3β,16α,20S,25α)-form, 3-O-β-_(D)-Glucopyranoside16,28-Secosolanida-5,22(28)-diene-3,16-diol, _(9Cl); 36506-65-7(3β,16α,20S,25α)-form, 16-Ac16,28-Secosolanida-5,22(28)-diene-3,16-diol, _(9Cl); 30511-97-8(3β,16α,20S,25α)-form, 16-Ac, 3-O-β-_(D)-glucopyranoside16,28-Secosolanid-5-ene-3,16-diol; 65027-01-2 (3β,16α,22R,25S)-form16,28-Secosolanid-5-ene-3,16-diol; 65027-00-1 (3β,16α,22S,25S)-form16,28-Secosolanid-5-ene-3,16-diol; 36069-45-1 (3β,16α,22S,25S)-form,16-Ac Shinonomenine 70598-84-4 Solanidine, O-β-_(D)-Galactopyranoside511-37-5 Synaine Tienmulilmine Tienmulilminine Veracevine;3-(Z)-2-Methyl-2-butenoyl 62-59-9 Veracintine 33596-06-4 Veracintine;3-O-β-_(D)-Glucopyranoside 67006-43-3 Veracintine; 3-O-α-_(L)-Rhamnoside110934-18-4 Veraflorizine 70598-85-5 Veragenine Veralbidine VeralinineVeralinine; Stereoisomer, 3-β-_(D)-Glucopyranoside 58078-63-0Veralkamine 17155-31-6 Veralkamine; O,O-Di-Ac 195244-85-0 Veralkamine;5α,6,12,13-Tetrahydro 17155-36-1 Veralobine 6242-49-5 Veralodine41787-59-1 Veralodisine 52617-23-9 Veralodisine;3-O-β-_(D)-Glucopyranoside 56598-27-7 Veralosidinine 52389-14-7Veramanine 182816-87-1 Veramarine 4565-85-9 Veramine 21059-48-3Veraminine Veramitaline 313697-00-6 Veranovine Verareine Veratramine60-70-8 Veratramine; O³-β-_(D)-Glucopyranoside 475-00-3 Veratramine;23-Deoxy Veratramine; 20-Epimer Veratramine; 20-Epimer,O²³-β-_(D)-glucopyranoside 148440-62-4 Veratra-5,11,13-triene-3,23-diol;148440-63-5 (3β,22S,23R,25S)-form, 23-O-β-_(D)-GlucopyranosideVeratrenone 55839-66-2 Verazine 14320-81-1 Verazine; 22S,N-Dihydro17463-47-7 Verazine; 22S,N-Dihydro, 3-O-β-_(D)-glucopyranoside128351-76-8 Verazine; 12β-Hydroxy, 22S,N-dihydro 164178-47-6 Verazine;20-Epimer 145033-50-7 Verdine 73667-53-5 Verine Vertaline B 118985-28-7Vertaline B; 16-Deoxy 91423-75-9 Vertaline B; 16-Deoxy,3-O-β-_(D)-glucopyranoside 128351-77-9 Zygadenilic acid δ-lactoneZygadenilic acid δ-lactone; O¹⁶-Angeloyl Zygadenine; O³—Ac 2777-79-9Zygadenine; O³-(R)-2-Methylbutanoyl Zygadenine; O³-Angeloyl 67370-02-9Zygadenine; O³-Angeloyl, β-N-oxide 313677-61-1 Zygadenine;O³-(3,4-Dimethoxybenzoyl) 31329-58-5

In one embodiment, the alkaloid is an alkaloid capable of being producedfrom a Veratrum or Amianthium cell. In another embodiment, the alkaloidis an alkaloid capable of being produced from a Veratrum cell. In oneembodiment, the alkaloid itself possesses therapeutic activity, or itcan be modified to yield bioactive compounds. In a preferred embodiment,the alkaloid contains a C-nor-D-homo-[14(13->12)-abeo] ring. In anotherpreferred embodiment, the alkaloid is cyclopamine or jervine.

The method includes culturing alkaloid-producing cells to produce one ormore alkaloids, as defined above. The term “alkaloid-producing cells”refers to any cells capable of producing one or more alkaloids under atleast one set of culture conditions. In preferred embodiments,“alkaloid-producing cells” refers to cells which produce one or morealkaloids in a detectable amount under the culture conditions of theembodiment.

In the alkaloid production methods described herein, the cell culturecomprises cells of the family Liliaceae. In one preferred embodiment,the cell culture comprises cells of the genera Veratrum and/orAmianthium. In a more preferred embodiment, the cell culture comprisesV. californicum cells. The cells in culture can be the same as ordifferent from one another. For example, the cells can be from one ormore genera, species, variants, or strains. The cells can benaturally-occurring, or they can be hybrids or genetically alteredcells. Exemplary plant cells include, but are not limited to, thoselisted in the table below:

TABLE 2 Exemplary cell species useful for alkaloid production ExemplarySub-Species/ Exemplary Genus Exemplary Species Varieties Amianthiummuscitoxicum Fritillaria camtschatcensis Schoenocaulon officinaleVeratrum album ssp. album, ssp. oxysepalum, ssp./var. lobelianum, var.Grandiflorum Veratrum calfornicum var. calfornicum, var. CaudatumVeratrum eschholtzii Veratrum fimbriatum Veratrum grandiflorum Veratrumlobelianum Veratrum maackii Veratrum nigrum var. Ussuriense Veratrumoblongum Veratrum officinalis Veratrum oxysepalum Veratrum patulumVeratrum sabadilla Veratrum stamineum Veratrum stenophyllum Veratrumtaliense Veratrum viride (viridae) var. Verabore Veratrum woodiiZygadenus gramineus Zygadenus paniculatus Zygadenus venenosus

The tissue used to initiate cell culture can be selected based on, forexample, ability to favor the production of one or more particularalkaloids.

The cell culture can be a callus culture. A callus is a substantiallyundifferentiated cell mass cultured on solidified medium. Methods forcallus formation and callus propagation are generally known in the art.In one embodiment, a callus is initiated by any viable part of themonocotyledonous plant, preferably immature embryos of themonocotyledonous plant. For example, cell culture initiation can includesurface sterilizing plant source material, e.g., by washing thoroughlywith clean water, using a disinfectant such as hypochlorite, usingwetting agents such as Tween or Triton, using antibiotics, and/or usingantifungal agents. The plant part can be used intact, or a portion of itcan be used, such as an embryo removed from a seed. Typically, the plantpart is placed on the surface of solidified medium and incubated in asterile environment for about 1-12 weeks, until a mass ofundifferentiated cells (the callus) grows in proximity to the plantpart. After establishing the callus culture, the cells are cultured in anutrient medium as described in further detail below. Theundifferentiated cells can be distinguished from embryogenic cells bythe presence of a vacuole because embryogenic cells are highlycytoplasmic and non-vacuolated.

For callus propagation, culture conditions including media components,pH ranges, carbon sources, nitrogen sources, macro-salts andmicro-salts, vitamins, and growth regulators are all described, forinstance, in Bringi WO 97/44476, incorporated in its entirety herein byreference. In one embodiment, callus propagation comprises using agelling agent, anti-browning agent, charcoal, and/or light/dark cycles.Gelling agents include, for example, agar, hydrogels, gelatin, andgelrite. Charcoal can be used for removing wastes and undesirableorganic compounds. An exemplary inoculum is about 0.01 to about 10 g/25ml. Subculturing techniques known in the art can be used for periodicserial transfer of portions of callus into a fresh source of nutrients.

The cell culture for producing alkaloids can be a suspension culture.The term “suspension culture” is used to describe structurallyundifferentiated cells that are dispersed in a liquid nutrient medium.It is understood that suspension cultures comprise cells in variousstages of aggregation. A range of aggregate sizes are encountered in thesuspensions with sizes ranging from tens of microns in diameter (singlecells or few-aggregated cells) to aggregates many millimeters indiameter, consisting of many thousands of cells. Generally, suspensionculture can be initiated using a culture medium that was successful incallus culture, without gelling agents, although optimized media forsuspension culture may differ from the optimum for callus of the samecell line. A cell culture may also be optionally derived from acryopreserved collection of cells.

Cryopreservation via 2-Step Method

In one embodiment, the invention provides a 2-step method for theinduction of desication tolerance in Veratrum plant cell suspensioncultures, as well as dehydration and cryoprotection steps prior tofreezing of the plant cell suspension cultures in cryogenic conditions.Cell suspensions of Veratrum plant cells typically contain many largecell aggregates, and alternative cryopreservation methods such as forexample vitrification are disadvantageous for use with Veratrum plantcell suspensions when the plant cell suspension cultures contain asignificant amount of highly aggregated plant cells, unless steps aretaken to minimize cell aggregation. Vitrification solutions have a veryhigh osmolarity, which can cause the cells on the outside of theaggregate to become injured by the vitrification solution before thevitrification solution penetrates the cells located in the internalportions of the cell aggregate. This phenomenon makes the timing ofexposure to vitrification solutions very difficult to determine, andthus undesirable for use with plant cells that are substantialaggregates. However, in one embodiment of the invention discussed below,cell aggregates are substantially eliminated thereby allowing the use ofvitrification methods.

Accordingly, the methods of the invention were developed as an effectivemeans of cryopreserving Veratrum plant cell suspensions with a highlevel of restoration of cellular function following thawing of thecryopreserved plant cells. In one embodiment of the invention, themethod provides greater than 50% recovery of viable cells followingthawing of the cryopreserved Veratrum plant cells using thecryopreservation techniques set forth herein. In another embodiment ofthe invention, the method provides greater than 75% recovery of viablecells following thawing of the cryopreserved Veratrum plant cells usingthe cryopreservation techniques set forth herein.

When cryopreservation is desired, Veratrum plant cell suspensioncultures that are to be cryopreserved are pre-cultured in liquid GM(IND64) media for about 7 to about 10 days. In a preferred embodiment ofthe invention, the plant cell suspension culture is a plant cellsuspension culture comprising undifferentiated cells. Cell suspensionsshould be transferred to fresh media every about 7 to about 10 days.Subsequently, the plant cell suspension cultures are transferred tosolidified GM (IND64) media comprising about 0.8% agarose to about 1%agarose and cultured in the dark for about 7 days to about 10 days at25° C.

TABLE 3 GM (IND64) Media* Component Amount Sucrose 20 g/L SH Macro (10X)100 ml/L SH Micro (1000X) 1 ml/L Iron Stock (50X) 10 ml/L SH Vitamins(50X) 20 ml/L Dicamba (10 mM) 1 ml/L *About 8 g/L or more of agarose isadded to the formulation to make solid media. Final pH of about 5.6.

In this embodiment following pre-culturing of the Veratrum plant cellson solid GM (IND64) media for a period of between about 7 to about 10days, the plant cells are cryopreserved using the 2-stepcryopreservation methods of the invention. Cryopreservation methods ofthe invention involve transferring the Veratrum plant cells into liquidGM media comprising from about 0.3M to about 0.5M sorbitol or sucrose,and culturing the Veratrum plant cells for about 16 hours to about 48hours on a rotary shaker (120 rpm). In a preferred embodiment of theinvention, the transferred Veratrum suspension plant cells are smallaggregates of plant cells.

In another embodiment of the invention, the transferred Veratrum plantcells are substantially small-aggregated through the use of, e.g., aBellco homogenizer and a Bellco Cellector™ Tissue Sieve (10 mesh/1910 μmand 20 mesh/860 μm) (Bellco Biotechnology, Vineland, N.J.).

The liquid GM media is removed and the Veratrum plant cells aresubsequently transferred into liquid Cryopreservation 1 media. Table 4provides the components of the Cryopreservation 1 media.

TABLE 4 Cryopreservation 1 Media Components Liquid GM media [IND64]Selected Sugar* [0.5M-1.0M] DMSO [5.0-10.0%] *By a selected sugar isintended a neutral sugar, an alcohol sugar, sucrose, maltose, trehaloseor glycerol. Neutral sugars include but are not limited to glucose,arabinose, xylose, mannose, galactose, rhamnose or glucuronic acid.Alcohol sugars include but are not limited to maltitol, sorbitol,xylitol, isomalt, lactitol, erythritol or mannitol.

In a preferred embodiment of the invention, the Cryopreservation 1 mediacomprises about 6% DMSO. The Veratrum plant cells are incubated in theCryopreservation 1 media for no less than about 2 hours to no greaterthan about 4 hours on ice, after which time 2 ml volumes of the plantcell suspension are transferred in Cryopreservation 1 media tocryo-vials. The transferred plant cell suspension cryo-vials are thencooled to from 0° C. to −40° C. at a rate of −0.33 to −1° C. per minute.Following cooling, the cryo-vials are submerged in liquid nitrogen andstored in a liquid nitrogen tank. FIGS. 5-7 demonstrate recovery ofcryopreserved Veratrum plant cell culture that may be obtained byperforming a cryopreservation technique consistent with the teachingsprovided herein.

Cryopreservation via Vitrification

In another embodiment of the invention, following pre-culturing of theVeratrum plant cells on solid GM (IND64) media for a period of betweenabout 7 to about 10 days the plant cells are cryopreserved usingvitrification techniques. In this embodiment of the invention. Veratrumplant cells are transferred into liquid GM (IND64) media comprisingabout 0.3M to about 0.5M sorbitol or sucrose, and the plant cells arecultured for about 16 hours to about 48 hours on a rotary shaker (120rpm). In a particularly preferred embodiment of the invention, thetransferred Veratrum suspension plant cells are small-aggregated plantcells produced using a Bellco homogenizer and a Bellco Cellector™ TissueSieve (20 mesh/860 um) (Bellco Biotcchnology, Vineland, N.J.).

Next, the liquid GM (IND64) media is removed and 10-30% w/v of freshVeratrum plant cells are subsequently transferred into Cryopreservation2 media. The composition of Cryopreservation 2 media is provided inTable 5. If necessary, the pH is adjusted to between about pH 5-7.

TABLE 5 Cryopreservation 2 Media Components Selected Sugar* [0.2M-0.5M]Permeating Agent [DMSO 0-25% and/or Ethylene Glycol 0-2M] SelectedTrisaccharide** [50-200 mM] *By a selected sugar is intended a neutralsugar, an alcohol sugar, sucrose, maltose, trehalose or glycerol.Neutral sugars include but are not limited to glucose, arabinose,xylose, mannose, galactose, rhamnose or glucuronic acid. Alcohol sugarsinclude but are not limited to malitiol, sorbitol, xylitol, isomalt,lactitol, erythritol or mannitol. **By a selected trisaccharide isintended melezitose, panose, raffinose, kestose or lactosucrose.Preferably, the Veratrum plant cells are incubated in theCryopreservation 2 media (cell density = 20%) for about 2 hours to about4 hours at about 4° C., after which time the Cryopreservation 2 media isremoved. About one part of the Cryopreservation 2 media-treated Veratrumplant cells are weighed and added into each cryo-vial. About 5 parts byweight of Cold Cryoprotectant Solution is added into each cryo-vial andthe cells are chilled at 0° C. (on ice) prior to submersion in liquidN₂. The composition of Cold Cryoprotectant Solution is provided in Table6. Immediately following this incubation, the cryo-vial is submerged inliquid nitrogen.

TABLE 6 Cold Cryoprotectant Solution Media Components Selected Sugar*[1.0-2.0M] Divalent Cation [5.0-10.0 mM] Permeating Agent [DMSO 0-50% orEthylene Glycol 0-10M] *By a selected sugar is intended a neutral sugar,an alcohol sugar, sucrose, maltose, trehalose or glycerol. Neutralsugars include but are not limited to glucose, arabinose, xylose,mannose, galactose, rhamnose or glucuronic acid. Alcohol sugars includebut are not limited to malitiol, sorbitol, xylitol, isomalt, lactitol,erythritol or mannitol.

Thawing and Recovery

The present invention also provides methods for the thawing and recoveryof cryopreserved Veratrum plant cell suspensions. In one embodiment ofthe invention, cryovials comprising cryopreserved Veratrum plant cellsuspensions are thawed at a temperature range of about 37° C. to about42° C. in a water bath or other sustained temperature environment, withoccasional agitation or gentle stirring for 3 to 5 minutes or until thefrozen cells have thawed.

Following thawing and sterilization of the cryovials (typically byethanol exposure), if the 2-step method is used, the contents of thecryovials are passed through a suction filter to remove thecryopreservation solution, and the cells on the filter paper aretransferred to solid GM (IND64) media, and incubated at 25° C. in thedark for 16-24 hours. Thawed cells are transferred to fresh media aboutevery 7 days until 1-2 grams of biomass are produced. If thevitrification method is used, the contents of each thawed and sterilizedcryo-vial is poured/diluted in 10 mls of media containing about 5-10 mMdivalent cations and about 0.1-1.0 M of a selected sugar for 10 minutes,then passed through a suction filter to remove the solution, and thecells on the filter paper are transferred to solid GM (IND64) media andincubated at 25° C. in the dark for about 16 to about 24 hours. Then,the filter paper with cells is transferred to fresh solid GM (IND64)medium and then transferred every 7 days to fresh GM medium at 25° C. inthe dark for restoration of cell functions.

Cell Culture: Nutrient Medium

The methods of the present invention include a step of culturing thecells in a nutrient medium. The method can also include more than onestep of culturing the cells in a nutrient medium. The term “nutrientmedium” means a medium that is suitable for the cultivation of plantcell callus and/or suspension cultures. The term “nutrient medium” isgeneral and encompasses both “growth medium” and “production medium.” A“growth medium” is a nutrient medium that favors growth of culturedcells. In preferred embodiments, the growth medium provides a growthincrease of at least 50% in one week. A “production medium” is anutrient medium that favors the production of one or more alkaloids.While growth can occur in a production medium, production can take placein a growth medium, and both growth and production can take place in asingle nutrient medium, a production medium favors the production oftarget compounds relative to the growth medium. The method describedherein can include one or more steps of culturing the cells in a growthmedium and/or one or more steps of culturing the cells in a productionmedium.

In the context of non-growth-associated secondary metabolites, aproduction medium preferably has a) an increased level of sucrose orother carbon course, and/or b) a reduced level of an inorganic componentsuch as nitrate, ammonium, phosphate, and/or potassium, and/or c) adifferent calcium level as compared to the growth medium. See U.S. Pat.No. 4,717,664. One of ordinary skill in the art recognizes that cellgrowth is generally favored by a balanced or relatively low ratio ofcarbon to inorganic components such as nitrogen and phosphate, whilecell growth is limited by a relatively high ratio of carbon to inorganiccomponents. Accordingly, the production medium may utilize growthlimiting conditions, e.g., a high ratio of carbon to inorganiccomponents, to promote alkaloid production as opposed to cell growth.See, e.g., Sakuta M. & Komamine A., “Cell Growth and accumulation ofSecondary Metabolites”, Cell culture and Somatic Cell Genetics ofPlants, Chapter 5, Vol. 4, pp. 97-114 (1987): Majerus F. & PareilleuxA., “Alkaloid accumulation in Ca-alginate entrapped cells ofCatharanthus roseus: Using a limiting growth medium”, Plant Cell Reports(1986) δ: 302-305. Nutrient media can be based on Murashige and SkoogBasal Salts (MS) or Schenk and Hildebrandt Basal Salts (SH)(Sigma-Aldrich). Exemplary production media (PM) include, but are notlimited to, media containing a salt base (e.g., MS or SH), plusmacronutrients, micronutrients, vitamins, 5% sucrose, 100 μM methyljasmonate (MJS), and 20 μM Dicamba. Exemplary growth media include, butare not limited to, media containing a salt base (e.g., MS or SH), plus2% sucrose, and 10 μM Dicamba.

In one embodiment, the cells are first cultured in a growth medium, andthen the cells are cultured in a production medium that is differentfrom the growth medium. When cells are transferred from a growth mediumto a production medium, the production medium preferably has a higherlevel of carbon source and/or C:N ratio, e.g., a higher concentration ofa saccharide. The production medium also preferably comprises sources ofinorganic or organic nitrogen such as an amino acid. Other components ofthe nutrient medium can be introduced into the culture after the cellsand medium are first contacted. In one embodiment, these ingredients,such as additional saccharide, are supplied in a feed streamintermittently or continuously as needed. Of course since the alkaloidproducts contain nitrogen, it is desirable to provide adequate nitrogento sustain and improve the accumulation of the desired products.

The desired effect, e.g., growth or production, can be achieved bymanipulating other reaction conditions including, but not limited to,temperature, pH, and light/darkness; by manipulating the mediaconditions by adding, removing, or changing the concentration of one ormore nutrients or other agents; or by manipulating any combination ofthese conditions. In some preferred embodiments, the desired effect isachieved by manipulating the medium. In particular, suspension culturesproducing alkaloids are capable of rapid growth rates and high celldensities when suitable nutrients and reaction conditions are used. Oneof ordinary skill in the art can readily incorporate, modify, andmanipulate media conditions in view of the guidance provided herein toachieve optimum performance, which may be expected to vary between celllines.

The alkaloids of the present invention are secondary metabolites thatare produced through a series of many enzymatic steps, requiringcoordinated action of many different enzymes to produce and sequentiallymodify precursors that are ultimately converted into target secondarymetabolites. At the same time, secondary metabolite production will belowered if other enzymes metabolize precursors of the desiredmetabolite, draining the precursor pools needed to build the secondarymetabolites. Stimulators of particular enzymes or inhibitors of otherenzymes may therefore enhance the rate and/or final yield of secondarymetabolites in culture of particular cell lines. In addition tonutrients typically employed in plant cell culture, other ingredientscan be included to improve alkaloid production. In one embodiment, thenutrient medium includes at least one component selected fromenhancement agents, elicitors, stimulants, precursors, inhibitors,growth regulators, heavy metals, and ethylene inhibitory compounds.

Adding one or more enhancement agents to the cell culture may improvealkaloid production. Enhancement agents include, but are not limited to,anti-senescence agents, agents affecting either the biosynthesis oraction of ethylene, plant growth regulators, precursors, inhibitors ofcompeting reactions for precursors or the desired products, elicitors,jasmonate and related compounds of the 12-oxo-phytodienoic acid pathway,compounds having auxin-like activity and precursors thereof, andcompounds having cytokinin-like activity. Adding precursors into theculture medium including, but not limited to, cholesterol and/or acetateand/or salts thereof may improve the biosynthesis rate and/or finalyield. Such precursor feeding might be combined with other factors like,but not limited to, inhibitors of undesired biosynthetic pathways and/orstimulation of the desired biosynthetic pathway e.g. by light.

The production of indole alkaloids in Catharanthus roseus cell culturesis known to be suppressed by added auxin-like compounds; likewise, inthe culture medium for alkaloid production, omitting or lowering thecontent of auxin-like substances improved production. Improvement ofalkaloid production by selected enhancement agent(s) can beexperimentally confirmed.

The rate of alkaloid production is determined not by a singlerate-limiting step, but by a complex interaction between a plurality oflimiting factors. Relief of any one of the limiting factors will enhancealkaloid production, although the magnitude of the enhancement willdepend on particular culture conditions, which determine the relativelimiting effects of other steps in alkaloid production once a particularlimitation has been relieved. Culture conditions that affect theinteraction between various limiting factors include: the genetic makeup of the cells: the composition of the culture medium; and the gaseousenvironment, temperature, illumination, and process protocol. Theenhancement agent(s) added to a particular culture will usually beselected in view of the limiting factors in that culture, which may bedetermined empirically by comparing the effects of individualenhancement agents as set forth herein. Typical quantities ofenhancement agents for cell culture are known in the art.

Elicitors include, but are not limited to, jasmonic acid, methyljasmonate, natural or synthetic jasmonates, tuberonic acid, cucurbicacid, coronatine, indanoyl amides such as 6-ethyl-indanoyl isoleucine,alkanoic acids, 12-oxo-phytodienoic acid, systemin, volicitin, andcompounds related to any of these exemplary elicitors. Elicitors alsoinclude, but are not limited to, oligosaccharides, e.g.,oligosaccharides from plants, fungi, or microbes; chitosan; chitin;glucans; cyclic polysaccharides; preparations containing cellularmaterial from bacteria, fungi, yeasts, plants, or insects; materialcontained in insect saliva or secretions; inhibitors of ethylenebiosynthesis or action in plants, especially silver-containing compoundsor complexes, cobalt, and aminoethoxyvinylglycine.

Elicitors especially useful for the production of the Veratrum alkaloidsinclude jasmonic acid-related compounds such as jasmonates, cucurbates,and tuberonates. Indanoyl amides are also useful elicitors. Heavy metalssuch as cadmium, vanadium and silver are useful in salt or complex form.Chitin, chitosans (especially chitosan glutamate), N-acetyloligosaccharides, pectic polysaccharides, fungal glycans (containing 5or more sugars), fungal glycoproteogalactans, sphingolipid elicitors,pectic polysaccharides, and arachidonic acid are also useful elicitors.

Exemplary growth regulators and inhibitors include, but are not limitedto, those disclosed in Bringi WO 97/44476. These can be used singly orin any combination. In general, plant growth regulators include a widevariety of substances readily known from the general plant literature.Of these, growth regulators particularly suitable for Veratrum culturesinclude, but are not limited to, auxin and/or cytokinin-like compounds,e.g., indoleacetic acid, indolebutyric acid, naphthalene acetic acid(NAA), phenoxyacetic acid and halogen substituted phenoxyacetic acids,picloram, dicamba, benzylaminopurine, kinetin, zeatin, thidiazuron, andindole acetic acid. Amino acids include any natural or synthetic aminoacid utilized by the cells such as glutamine, glutamic acid, andaspartic acid.

Other compounds including, but not limited to, alpha amino isobutyricacid (which simulates rol C-induction) and/or alphaDL-di-fluormethylornithine (which can induce a similarphenotype/biochemical situation to that induced by RI-T-DNA) might beadded to the culture medium in order to reduce unrelated compoundsand/or in order to improve product yields.

It is also desirable to reduce or avoid oxidative browning in the cellculture. Thus, anti-browning agents may be added to inhibit theoxidation of phenolic exudates. Preferably, the cells in culture areundifferentiated cells, which are less susceptible to browning thanembryonic cells.

Suggested concentration ranges for these medium components are providedbelow, but routine optimization may demonstrate preferred conditionsoutside these ranges for particular cell lines and/or particular cultureconditions. All concentrations refer to average initial values in theextracellular medium after addition. Concentrations in feed solutionsand therefore, locally, concentrations in contact with the cells couldbe higher than that indicated. Elicitors, such as jasmonic acid-relatedcompounds, can be used at doses of about 0.01 μmol/L to about 1 mmol/L,preferably about 1 μmol/L, to about 500 μmol/L. Preparations containingcellular material can be added based on the concentration of a specificconstituent of the preparation or as some fraction of the culturevolume. Heavy metals and ethylene inhibitors such as silver salts orcomplexes, can be used at concentrations up to about 1 mmol/L,preferably about 0.01 μmol/L to about 500 μmol/L. Other inhibitors ofmetabolic pathways can be used at concentrations of about 1 μmol/L toabout 5 mmol/L. Aromatic compounds including a methylenedioxyfunctionality can be included at concentrations of about 0.1 μmol/L toabout 5 mmol/L, preferably about 1 μmol/L to about 2 mmol/L. Growthregulators such as compounds with auxin-like activity; compounds withcytokinin-like activity; compounds with giberrelic acid-, absicic acid-,or brassinosteroid-like activity, can be used at concentrations of about0.001 μmol/L to about 2 mmol/L, preferably about 0.01 μmol/L to about 1mmol/L. Precursors, such as amino acids or terpenoid precursors, can beused at concentrations of about 1 μmol/L to about 20 mmol/L, preferablyabout 10 μmol/L to about 10 mmol/L. Using these suggested concentrationsas a guide, routine optimization can discern which component, orcombination of components, and which concentrations, includingconcentrations beyond the suggestions above, are useful to maximizealkaloid production.

Cell Culture: Other Reaction Conditions

As described above, the cells can be cultured under conditions to favorproduction and/or growth. In addition to modifying the mediumcomponents, other reactions conditions can also be modified to obtainthe desired result. For instance, the temperature range can be adjusted.Preferred temperatures include about 0° C. to about 35° C., morepreferably about 15° C. to about 35° C., and even more preferably about20° C. to about 30° C. Likewise, one can alter the periods of thelight/dark cycle, or one can apply within a production cycle a firstperiod of time—more than 24 hours—in the dark followed by a secondperiod of time—more than 24 hours—of illumination (preferably about 10μmol photons m⁻²s⁻¹ to about 250 μmol photons m⁻² s⁻¹). This proceduremight be applied once or twice, or it might be applied several times ina repetitive manner.

The levels of gases such as oxygen, carbon dioxide, and ethylene can becontrolled to favor alkaloid production or to favor biomassaccumulation. Routine cultivation in lab scale cultivation vessels heldin an atmosphere of air, with typical closures such as sheets, plugs, orcaps result in dissolved oxygen levels below air saturation and levelsof carbon dioxide and ethylene higher than that present in atmosphericair. Thus routinely, carbon dioxide levels in the head-space of theculture are typically greater than about 0.03% v/v. However, theconcentrations of carbon dioxide and/or ethylene can be adjusted tofavor alkaloid production or to favor cell propagation. In oneembodiment, alkaloid production is favored by adjusting the carbondioxide level in the head-space or aeration stream to be about 0.1%(approximately 3-times atmospheric) to about 10%, preferably about 0.3%to about 7% in equilibrium with the liquid at the temperature ofoperation. In another embodiment, ethylene is less than about 500 ppmand preferably less than 200 ppm as measured in the gas phase inequilibrium with the liquid phase at the temperature of operation. Gasescan also be provided independently, e.g., the sources of oxygen andcarbon dioxide can be different.

Dissolved gases can be controlled by varying one or more of: theagitation rate, the composition of aeration gas, the supply rate of theaeration gas, the venting rate of the aeration gas, and the totalpressure in the cultivation vessel. Agitation rates can be controlled atabout 1 to about 500 per minute (rotations or oscillations of agitatorsor circulations of fluid). The supply rate of gas can be any rate thatis appropriate for achieving a dissolved gas concentration that isadequate or optimal for cell biomass accumulation or maintenance orproduct formation. Preferably, this rate is about 0.01 to about 10volumes of gas per volume of culture broth per minute and can besupplied directly into the culture liquid, or into a separate portion ofliquid that is subsequently mixed with the rest of the culture, or intothe head space of the culture, or into a device for contacting the gasspecies with the culture medium. In one embodiment, dissolved oxygenconcentrations are controlled at about 10% to about 200%, preferablyabout 20% to about 150%, of air saturation at the operating temperature.Of course, it is possible that for various operational reasons, e.g.,temporary reduction in aeration, the dissolved oxygen level could be aslow as zero for periods of time ranging from a few minutes to severalhours. Specific useful combinations of oxygen, carbon dioxide, andethylene, outside these ranges may be discovered through routineexperimentation and are considered to be within the scope of thisinvention.

Cell Culture: Process Operations

The operating mode for a plant cell culture process refers to the waythat nutrients, cells, and products are added or removed with respect totime (Payne et al. 1991). Ingredients provided to the cells can beprovided in a number of different ways. Ingredients can be added in aparticular stage of growth such as lag, exponential, or stationary. Allingredients can be provided at once and then after a suitable period oftime, product can be recovered. Alternatively, not all ingredients canbe provided all at once. Rather one or more of them can be provided atdifferent times during the cultivation. Further, the additions can bediscontinuous or staggered as to the time of initial contact and theduration of such provision can vary for different ingredients.Ingredients can be provided in a plurality of parts. One or moreingredients can be supplied as part of solutions separately contactedwith the cell culture or portions thereof. Portions of the culture canbe removed at any time or periodically and used for cryopreservation,further cell propagation, production, and/or recovery. Suchcell-containing portions can be exposed further to nutrients or otheringredients as desired. Exemplary subculture procedures are describedherein.

In one embodiment, medium containing nutrients or other ingredients canbe added to replenish a portion or all of the removed volume. Thereplenishment (dilution) rate (volumetric rate of addition divided bythe volume of liquid in the vessel) can vary between 0.1 times to 10times the specific growth rate of the cells. Portions of such removedmaterial can be added back into the original culture, for instance,cells and medium can be removed, a portion of the cells or medium can beused for product recovery and the remaining cells or medium can bereturned. The supply rate of ingredients to the culture or levels ofvarious ingredients in the culture can be controlled to advantageouslyproduce and recover the product. Separate portions of the culture can beexposed to ingredients in any of the foregoing modes and then combinedin proportions determined to be advantageous for production. Also thecell content of the culture can be adjusted to advantageously yieldproduct or propagate cells. Adjustment of cell content can beadvantageously combined with strategies for contacting with nutrients orother ingredients.

The culture method can include medium exchange. As documented forinstance in Bringi WO 97/44476, the removal of spent medium andreplenishment of fresh medium periodically. e.g., every few days, cansignificantly enhance total production, as well as increase the amountsof extracellular product. The stimulatory effects of medium exchange maybe due to removal of product in situ, which would prevent feedbackinhibition and product degradation. Such positive effects of in situproduct removal on secondary metabolite production and secretion insuspension cultures of unrelated plants have been documented by, amongothers, Robins et al., “The Stimulation of Anthraquinone Production byCinchona ledgeriana Cultures with Polymeric Adsorbents,” Appl.Microbiol. Biotechnol. 24: 35-41 (1986) and Asada et al., “Stimulationof Ajmalicine Production and Excretion from Catharanthus roseus: Effectsof Adsorption in situ, Elicitors and Alginate Immobilization,” Appl.Microbiol. Biotechnol. 30:475-81 (1989). The periodic removal of spentmedium incorporates the above advantages, and additionally, may serve tode-repress secondary biosynthesis by removing other inhibitorycomponents from the medium. In situ product removal can also be usedwithout medium exchange. For example, the product can be removed byresin absorption to stimulate further production.

The replenishment of fresh medium to cells undergoing activebiosynthesis may also enhance production by providing essentialnutrients that have been depleted. For example, Miyasaka et al. wereable to stimulate stationary phase cells of Salvia miltiorhiza toproduce the diterpene metabolites cryptotanshinone and ferruginol simplyby adding sucrose to the medium. Miyasaka et al., “Regulation ofFerruginol and Cryptotanshinone Biosynthesis in Cell Suspension Culturesof Salvia Miltiorrhiza,” Phytochemistry 25: 637-640 (1986). Presumably,biosynthesis had ceased due to carbon limitation in the stationaryphase. Using a periodic-medium-exchange protocol for the present culturemethod may provide similar benefits.

It is contemplated that the amount of medium exchanged, the frequency ofexchange, and the composition of the medium being replenished can bevaried in accordance with various embodiments of the invention. Theability to stimulate biosynthesis and secretion by medium exchange hasimportant implications for the design and operation of an efficientcommercial process in the continuous, semi-continuous, or fed-batchmode.

When all the nutrients are supplied initially, and the culture contentscomprising cells and product are harvested at the end of the cultureperiod, the operating mode is termed a “single-stage batch process.” Inpractice, some ingredients could be added first and others shortlyafterward for example, due to separate processing requirements fordifferent ingredients. When a batch process is divided into twosequential phases, a growth and a production phase, with the mediumbeing changed in between the two phases, the operating mode is termed a“two-stage growth/production batch process.” The method can include atransition from growth medium to production medium by an abrupt stepwisechange, or progressively by a series of steps, or by progressive,continuous change. In one extreme, the progressive change isaccomplished by progressive replacement of media of incrementallychanging composition. In another alternative, the progressive change isaccomplished by feeding one or more components of the production mediuminto the growth phase culture. This is one example of the fed-batchprocess. In a “fed-batch” operation, particular medium components suchas nutrients and/or one or more enhancement agents are supplied eitherperiodically or continuously during all or part of the course of aone-stage or a two-stage culture. A description of nutrients andenhancement agents may be found, for instance, in Table A or Tables 1and 2 of WO 97/44476. Additionally, a combination of abrupt andprogressive changes can also be employed. In one example, some portionof the nutrient medium can be changed abruptly while other componentsare slowly fed.

Using a fed-batch mode, it has been found that cells can be sustained ina productive state for a prolonged period, and in fact, thatproductivity of the cells can be enhanced. It will be apparent to theskilled artisan, that the composition of the feed may be varied toobtain the desired results, such as extension of the production phase toincrease product yield, or extension of the growth phase to achievehigher biomass density. Selection of suitable conditions to achieveoptimum productivity and performance is easily within the skill of theordinary artisan in view of the teachings described herein. Similarly,variations of other operating parameters, such as the timing andduration of the addition and the rate of the addition of the fed-batchcomponents, to achieve the desired results, are within the reach of theskilled artisan in view of the teachings described herein.

In one embodiment, a substantial portion, but not all, of the contentsof a batch culture is replaced by fresh medium for continued cell growthand production; this process mode resembles a “repeated draw and fill”operation and is termed a “semi-continuous process.” In anotherembodiment, the process is “continuous,” that is, fresh medium iscontinuously supplied, and effluent medium is continuously orrepetitively removed.

In one embodiment, the operation mode is “perfusion mode,” that is,cells are substantially retained within the reactor. In anotherembodiment, the process is “chemostat,” that is, cells are continuouslyremoved with the effluent medium.

Once initiated, a suspension culture can be further cultivated, eitherby separating the cells substantially from the medium (typically byfiltration) and then reintroducing a portion to a medium containingnutrients, or by transferring a volume of culture broth (cells andmedium) into a medium containing nutrients, or by allowing the cells tosettle followed by removal of any portion of medium already present andreintroducing nutrient-containing medium. When cells are separated andtransferred to a different nutrient-containing medium, the transferredamount can be about 0.3% to about 30%, preferably about 1% to about 25%,on a fresh weight basis. Note that as the cells acclimate and/or grow,this fraction may change. When cells and media are transferredvolumetrically, the ratio of the transferred volume to the final volumecan be from about 1% to substantially all of the volume. In this case,fresh nutrients can be supplied in a concentrated form, resulting inonly a small volume increase. The culture can thus be divided intoportions. In one embodiment, each portion is optionally used foralkaloid production. Each portion can, but need not, be cultured underthe same conditions as one another or as the original culture. Theculture duration is preferably 2-50 days, 2-15 days, 5-10 days, or about7 days. The duration of growth can be extended by supplementing apartially depleted medium with nutrients.

Alkaloid Recovery

Alkaloids can be recovered from the entire culture or any portion ofculture (medium only, cells only, or an amount of cells and mediumtogether). Cell material can be lyophilised in advance to the extractionprocedure. Other methods known in the art can be used in order toprepare cell material and/or medium for the appropriate extractionmethod. Alkaloids can be recovered by any method known in the artincluding, but not limited to, extraction using a non-aqueous polarsolvent, extraction by using an acid medium, extraction by using a basicmedium, recovery by resin absorption where the resin is either inside oroutside of the culture vessel. Alkaloids can be recovered at any timeduring the cultivation or after the completion of the culture period.

Although particular features may be described with respect to particularembodiments, each feature can be used independently or in combinationwith any other feature described herein to increase the production ofVeratrum alkaloids in cultures of Liliaceae. To further articulate theinvention described above, we provide the following non-limitingexamples.

EXAMPLES Example 1 Cultivation of Veratrum Cells

Veratrum cell cultures are initiated from any suitable part of theVeratrum plant using accepted techniques of plant cell culture.Substantially undifferentiated cells are propagated on solidified orliquid nutrient medium under the following conditions, except that cellscultivated on solidified medium do not require agitation. Veratrum cellsare cultivated at 20-30° C., at pH 4-7, in darkness, using agitation tomix the culture, and providing oxygen and other gases and ventilation bycontacting oxygen-containing gas with the cell suspension. The oxygen ismaintained at 10%-150% of air saturation at the operating temperature,and the carbon dioxide is maintained at higher than 0.05%. The level ofoxygen and other gases is controlled by adjusting the agitation,pressure, composition of gas, ventilation rate, and/or feed rate of thegas.

The medium contains components capable of supporting Veratrum cellgrowth. For example, the medium can contain sugar (about 1-100 g/L) andcumulative nitrogen from one or more sources (about 1-100 mmol/L). Themedium can also contain growth regulators such as auxin and/orcytokinin-like compounds. e.g., naphthalene acetic acid (NAA),phenoxyacetic acid and halogen substituted phenoxyacetic acids,picloram, dicamba, benzylaminopurine, kinetin, zeatin, thidiazuron,and/or indole acetic acid. The medium can optionally contain otherVeratrum alkaloids, amino acids such as glutamine, a source of silverion, e.g., silver nitrate or silver thiosulfate, or other ingredientcapable of affecting ethylene biosynthesis or action. The inoculumconcentration of Veratrum cells can be about 10 g fresh cell weight/L toabout 300 g fresh cell weight/L. The medium can also contain one or moreelicitors such as an indanoyl amide, ajasmonic acid-related substance,or another elicitor.

These medium components can be added at the beginning of the culture,after the exponential growth phase, or intermittently throughout theculture period. Medium components can be added at once or at differenttimes during the cultivation and can be fed continuously orintermittently. The ingredients can be added either before or after theinclusion of plant cells in the culture broth. Further, nutrients orother ingredients can be added during cultivation. Optionally, themedium can be changed after a suitable period of cultivation whereby thecells are freshly exposed to a medium containing similar ingredients asdescribed above. Such changes include changing the amount of one or moreof sugars such as glucose, fructose, sucrose, and maltose, and/orchanging the amount of one or more nitrogen sources such as nitrate,ammonium, amino acids, casamino acids.

After a period of cultivation, the culture is harvested and the levelsof Veratrum alkaloids are quantified using high performance liquidchromatography (HPLC), and specific Veratrum alkaloids are identifiedusing liquid chromatography-photodiode array-mass spectrometer(LC-PDA-MS). Veratrum alkaloids can be recovered from these cultures byappropriate extraction and purification procedures.

Example 2 Detection and Quantification of Veratrum Alkaloids

This example provides an appropriate analytical method for quantifyingcyclopamine, jervine, and related steroidal alkaloids in Veratrum sp.cultures. The initial screening was performed by liquidchromatography-photodiode array-mass spectrometer (LC-PDA-MS). Thistechnique is highly valuable in the identification of compounds incomplex mixtures, especially when using some specialized massspectrometric experiments like multiple reactions monitoring (MRM) andsingle ion monitor (SIM). This targeted approach is suitable forinvestigating the production of one or more target compounds in cellculture.

In the MRM experiments, QI scan (full scan, first quadrupole) and MSMSscan (fragment and productions are obtained) of an analyte is carriedout. Then specific fragmentation products of the protonatedpseudo-molecular ions (M+H⁺) are selected, based on their initial scan.Instrument optimization is then performed to achieve a more sensitivedetection based on the selected ion pairs. The selection method hasproven to be accurate, precise, and sensitive at the μg/L level.

The MRM experiments using a triple quadrupole instrument were designedto obtain the maximum detection sensitivity for target compounds. Thistype of mass spectrometric experiment is widely used for detecting andquantifying drug and drug metabolites in the pharmaceutical industry.

Using the above techniques, it is possible to predict the precursor m/zand a fragment m/z (MRM transition) for many common metabolites of adrug molecule once the structure and molecular weight of the moleculeare known. These MRM experiments can be used to screen for specificmetabolites, and trigger a dependent product ion scan to confirm themetabolite structure. The same principle can be applied to study theproduction and optimization of target molecules in a cell culture if themolecular weight and structure of the target molecule are known. Thesehighly sensitive MRM experiments were used to trigger dependentacquisitions of product ion scans (MS/MS). Cyclopamine and jervine weredetected in Veratrum sp. plant cell cultures using this method.

General Methodology:

The following methodology was used for each Example, except where noted.

Equipment and Materials:

Reagents: acetonitrile (HPLC grade), methanol (HPLC grade), CHCl₃, andglacial acetic acid (all purchased from VWR)

Standards: Cyclopamine, jervine, and/or tomatidine (Calbiochem).

Equipment: Quattro Premier triple quadrupole mass spectrometer(Micromass Ltd.), Acquity UPLC system (Waters), BEH C₁₈ column (1.7 μm,2.1×50 mm, Waters)

Standard Preparation

1 mg alkaloid (e.g., jervine, cyclopamine, tomatidine) was individuallydissolved in acidic methanol (0.1% acetic acid v/v) to a final volume of10 mL. The standard solution was sonicated for 1 min.

Sample Preparation:

Each Veratrum sample was crushed in a glass vial, and acidic methanol(20 mL, 1% formic acid) was added. The sample was sonicated for 2 hrs,then left in the dark overnight. The vial was centrifuged, and itssupernatant removed. The supernatant was concentrated in a rotaryevaporator, and the resulting extract was reconstituted in acidicmethanol (1% formic acid v/v).

Liquid Chromatographic Separation:

Separation was performed on a Waters Acquity UPLC system equipped with aBEH C₁₈ column (1.7 μm, 2.1×50 mm). Twenty microliter (20 μL) aliquotswere injected using an auto sampler. Solvent A was 0.05% formic acid inde-ionized water, and solvent B was HPLC grade acetonitrile with 0.05%formic acid.

Separation was achieved by an initial hold at 77% A for 1 minute, alinear gradient from 77% A to 100% B in 4.5 minutes, followed by holdingat 100% B for 0.1 minute. During the analysis, the flow rate was 350μL/min, and the column was kept at 35° C.

Mass Spectrometry:

All mass data was acquired on a Quattro Premier triple quadrupole massspectrometer (Micromass Ltd.). The mass spectrometer was operated underMassLynx V4.0 software. For the samples investigated, the instrument wasoperated in positive mode electrospray with capillary voltage of 3.50kV, a desolvation temperature of 120° C., a source temperature 120° C.,and sample cone voltage of 45 V. (For the exemplary detection ofcyclopamine and jervine, see Example 4, FIG. 3.)

Example 3 Production of Cyclopamine from Callus Culture

Seeds from Veratrum californicum were surface sterilized using standardtechniques and cultivated on agar-solidified growth media. In someinstances, the seeds were placed on water soaked filters for 1 to 3months, then moved to water/agar (0.5-1% agar w/v) combination for 2 to4 weeks. The growth media included:

MS salts, 2% w/v sucrose, and 10 micromol/L Picloram;

MS salts, 2% w/v sucrose, and 10 micromol/L Dicamba;

MS salts, 2% w/v sucrose, and 10 micormol/L alpha-naphthalene aceticacid (NAA).

Prior to initiation of suspension culture (described in Example 4),calli were initiated and then cultivated on solidified medium containingMS salts, 2% sucrose, and 10 μM Dicamba. Callus transfers were performedperiodically about every 1-8 weeks depending on callus growth.

Approximately 50 mg samples of calli were crushed in a glass vial andextracted three times, each with 2 ml acidic methanol and CHCl₃ mixture(1:1 v/v) for 60 minutes in a sonicating bath. Each time, the vial wascentrifuged and supernatant was removed. The combined supernatant wasconcentrated in a centrifuge evaporator (Genavac). The resulted extractwas reconstituted in chloroform (CHCl₃) and methanol (MeOH) mixture to afinal volume of 0.4 mL.

Chromatographic separation was achieved as described in Example 2 exceptwe used an initial hold at 95% A for 1 minute, a linear gradient from95% A to 100% B in 10 minutes, followed by holding at 100% B for 1minute. The flow rate was 0.3 mL/min.

For the mass spectrometry analysis, each liquid stream was sampled for0.1 s with mass spectra acquired from 150 to 1000 Da. The instrument wasoperated in positive mode electrospray with capillary voltage of 3.00kV, a desolvation temperature of 120° C., a source temperature 100° C.,and sample cone voltage of 45 V.

Cyclopamine was detected in both calli. See FIG. 1 and FIG. 2.

Example 4 Alkaloid Production from Suspension Culture

Suspension cultures were initiated by transferring about 1 g of callusmaterial to a 125 ml Erlenmeyer flask containing 25 ml of a liquidgrowth medium containing MS salts, 2% sucrose, and 10 μM Dicamba. Thissuspension culture was maintained in the dark by shaking at 120 rpm on a1′-throw shaker. The flasks were closed with an open cell silicone foamclosure to allow for gas transfer. The flasks were maintained in anatmosphere of air. The suspension cells from callus were transferred 5times at appropriate transfer intervals for a total of 45 days in liquidgrowth medium; the last cycle time was 11 days.

Suspension cells from four Veratrum californicum cultures wereinoculated into MS-PM (a Murashige and Skoog Basal Salts-basedproduction medium containing MS salts, macronutrients, micronutrients,vitamins, 5% sucrose, 100 μM methyl jasmonate (MJS), and 20 μM Dicamba)at a fresh weight inoculum concentration of 20% w/v. The cultures wereincubated in an atmosphere of air, in the dark, at 25° C. and shaking at180 rpm for 14 days.

Suspension cells were prepared for analysis according the generalmethodology (see Example 2), and the solvent from each 2 mL vial ofreconstituted extract was removed by stream of nitrogen. The sampleextracts were re-dissolved in 200 L of acidic methanol (1% formic acid)before analysis.

Data acquisition was performed with full scan and Multiple ReactionMonitoring (MRM) of selected fragmentation products of the protonatedpseudo-molecular ions (for cyclopamine m/z, M+H⁺, 412.69->321.65 and forjervine 426.58->313.54). Appropriate four-point calibration curves weregenerated for each targeted compound of interest by injecting 20 μl ofacidic MeOH (1% formic acid) solutions containing known amounts ofcyclopamine and jervine.

The identification of two alkaloids of interest-jervine andcyclopamine—was made by comparing their retention times, PDA UV spectra,MS spectra, and MS/MS spectra to those of known standards. Jervine andcyclopamine were detected in one cell culture (See FIG. 3 and FIG. 4).The concentration of cyclopamine in the extract was below the limit ofquantification, but above the limit of detection (3:1, signal to noiseratio). Cyclopamine was detected at the μg/L level.

Example 5 Alkaloid Production from Suspension Culture

Seeds from Veratrum californicum were surface sterilized and embryoswere extracted and placed on various solid media as described above inExample 3. After surface sterilization, these embryos were placeddirectly onto solid media containing SH salts, 2% w/v sucrose, and 10 μMDicamba. Cultivation on solid media was continued until callus ofundifferentiated cells was produced.

Portions of callus at a cell density of 5% (w/v) were placed in a liquidgrowth medium containing SH salts, 2% sucrose, and 10 μM Dicamba to forma suspension culture. The growth index for such suspension cultures wasabout 2 over a 7-day growth period. (Growth index is the ratio of thefinal fresh or dry weight to the corresponding initial fresh or dryweight over the duration of the culture period.) The cells wereseparated by filtration and then inoculated into MS-PM or SH-PM at astarting inoculation density of 20% w/v. Samples were incubated in anantmosphere of air at 85% humidity and 180 rpm in the dark at atemperature of 25° C. The cultures were sampled and analyzed after 8, 9,and 10 days of incubation. For one suspension cell culture of Veratrumcalifornicum, Veratrum alkaloid production was highest at day 9 in bothMS-PM and SH-PM. See FIG. 5A and FIG. 5B FIG. 6 and FIG. 7 show datafrom a repeated experiment using the same cell culture. Results foranother suspension cell culture of Veratrum californicum are shown inFIG. 8 and FIG. 9.

Cyclopamine and jervine were detected in each sample. Alkaloidproduction increased from day 8 onwards, then later declined toward day14.

Example 6 Cryopreservation of Veratrum Cells Using the Two-Step Method

Veratrum californicum strain VEACCSP-94 undifferentiated cells arepre-cultured in liquid GM (IND64) media for about 7 days. Followingpre-culturing, the undifferentiated cells are transferred to solid GM(IND-64) media plates comprising either about 0.8% or about 1.0%agarose.

Following pre-culturing, the Veratrum cells are transferred to liquid GM(IND-64) media comprising 0.3-0.5 M sorbitol and incubated at about 25°C. for about 24 hours. After incubation, the Veratrum cells are thentransferred to liquid Cryopreservation 1 media and incubated for about2-4 hours at between about 0° C. to 4° C.

The Veratrum cells are then cooled at a rate of about −0.33° C./minuteuntil the cells are cooled to about −40° C. The cooled cells areimmediately placed into liquid nitrogen and cryopreserved.

Example 7 Recovery of Thawed Veratrum Cells

The cryopreserved Veratrum cells of Example 6 are thawed from a frozenstate in a 42° C. water bath with occasional stirring until the cellsare no longer frozen. TTC cell viability tests are performed on thethawed cells to determine the cell viability of the thawed cells. TTCcell viability tests are well known to those of skill in the art. TheTTC test reveal that greater than 50% of the thawed cells were viable.

Example 8 Cryopreservation via Vitrification

Following pre-culturing of undifferentiated Veratrum plant cells onsolid GM (IND64) media for a period of about 7 days as set forthpreviously herein, the plant cells are cryopreserved using vitrificationtechniques. The cultured Veratrum plant cells are transferred intoliquid GM (IND64) media comprising about 0.5 M sorbitol, and the plantcells are cultured for about 32 hours on a rotary shaker (120 rpm). Thetransferred Veratrum suspension plant cells are small-aggregated plantcells produced using a Bellco homogenizer and a Bellco Cellector™ TissueSieve (20 mesh/860 um) (Belico Biotechnology, Vineland, N.J.).

Next, the liquid GM (IND64) media is removed and the Veratrum plantcells are subsequently transferred into Cryopreservation 2 media. Thecomposition of Cryopreservation 2 media is provided in Table 7.

TABLE 7 Cryopreservation 2 media Components Selected Sugar* [0.2 to0.4M] Selected Trisaccharide** [60 to 190 mM] Permeating Agent [EthyleneGlycol 0.5 to 1M] *By a selected sugar is intended a neutral sugar, analcohol sugar, sucrose, maltose, trehalose or glycerol. Neutral sugarsinclude but are not limited to glucose, arabinose, xylose, mannose,galactose, rhamnose or glucuronic acid. Alcohol sugars include but arenot limited to malitiol, sorbitol, xylitol, isomalt, lactitol,erythritol or mannitol. **By a selected trisaccharide is intendedmelezitose, panose, raffinose, kestose or lactosucrose. The Veratrumplant cells are incubated in the Cryopreservation 2 media (cell density= 20%) for about 3 hours at about 4° C., after which time theCryopreservation 2 media is removed. About one part of theCryopreservation 2 media-treated Veratrum plant cells are weighed andadded into each cryo-vial. About 5 parts of Cold Cryoprotectant Solutionis added into each cryo-vial and the cells are incubated at 0° C. (onice) for a short period. The composition of Cold Cryoprotectant Solutionis provided in Table 8. Immediately following this incubation, thecryo-vial is submerged in liquid nitrogen.

TABLE 8 Cold Cryoprotectant Solution Components Selected Sugar*[1.0-2.0M] Divalent Cation [5.0-10.0 mM] Permeating Agent [EthyleneGlycol 2-5M] *By a selected sugar is intended a neutral sugar, analcohol sugar, sucrose, maltose, trehalose or glycerol. Neutral sugarsinclude but are not limited to glucose, arabinose, xylose, mannose,galactose, rhamnose or glucuronic acid. Alcohol sugars include but arenot limited to malitiol, sorbitol, xylitol, isomalt, lactitol,erythritol or mannitol.

Cryovials comprising cryopreserved Veratrum plant cells are thawedfollowing cryopreservation at a temperature of about 40° C., in a waterbath or other sustained temperature environment, with occasionalagitation or gentle stirring for about 4 minutes or until the frozencells have thawed.

Following thawing and sterilization of the cryovials (typically byethanol exposure), the contents of each thawed and sterilized cryo-vialis poured/diluted in 10 mls of liquid media containing about 5-10 mMdivalent cations and 0.1-1.0 M sorbitol for 10 minutes, then passedthrough a suction filter to remove the solution, and the cells on thefilter paper are transferred to solid GM (IND64) media and incubated at25° C. in the dark for about 16 to about 24 hours. Then, the filterpaper with cells is transferred to fresh solid GM (IND64) medium andthen transferred every 7 days to fresh GM medium at 25° C. in the darkfor restoration of cell functions.

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled) 11.(canceled)
 12. (canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled)16. (canceled)
 17. A culture of undifferentiated Veratrum cells that iscapable of producing at least about 0.1 mg/L of one or more Veratrumalkaloids or a precursor of such an alkaloid or a mixture thereof.
 18. Asuspension culture comprising undifferentiated cells of Lilliacae andone or more Veratrum alkaloids in an amount of at least 0.1 mg/L. 19.Veratrum alkaloids or a precursor or a mixture thereof produced by cellculture of undifferentiated cells of the family Liliaceae
 20. An extractof a culture of undifferentiated Veratrum cells comprising one or moreVeratrum alkaloids, wherein the extract contains the one or morealkaloids in an amount of at least 0.1 mg/L.
 21. (canceled) 22.(canceled)